The general goal of the proposed research is to identify and define pathways that regulate homologous recombination as a DNA repair and DNA damage tolerance pathway in somatic cells. The fundamental approach is to use the budding yeast Saccharomyces cerevisiae as a lead organism and to establish novel mechanisms, which will be transferred by proof-of-principle experiments to humans to validate the general significance of the original paradigms. The Specific Aims are: 1. Determine the mechanism of anti-recombination and crossover regulation by Srs2. Srs2 is an anti- recombinase that acts by dissociating Rad51 from ssDNA. In addition, Srs2 has been demonstrated genetically to limit crossover formation. Sub-aim A: We will determine the mechanism of crossover regulation by Srs2. We will determine the roles of Rad55-Rad57 and PCNA and their post-translational modifications on the Srs2 anti-recombination and anti-crossover activity. Sub-aim B: We will extend these findings to humans and determine which proteins proposed to exert Srs2 function are involved in crossover control. 2. Determine the mechanism of activation of Mus81-Mms4. Mus81-Mms4 is a structure-selective endonuclease that processes HR-dependent joint molecules. We have reconstituted in vitro the Cdc5 (Polo kinase)-mediated activation of Mus81-Mms4 and developed an experimental plan to differentiate between different mechanistic models of direct activation of the Mus81-Mms4 catalytic activity. Sub-aim A: We will establish the mechanism of activation using genetic and biochemical approaches in yeast. Sub-aim B: We will conduct proof-of-principle experiments to show that also human MUS81-EME1 is directly activated by Polo- kinase-mediated phosphorylation.